Molecular Mechanisms of
Autophagy in Yeast
Nobel Lecture, December 7, 2016
by Yoshinori Ohsumi
Tokyo Institute of Technology, Tokyo, Japan.
S
cience is a system of knowledge that is gradually accumulated over many
years by society, but it is also an inherently human activity. I believe that
every scientist is a product of the era in which they live. So I’d like to start from
a brief introduction of my life, and then give a historical overview of my scientific work.
Early life
I was born in Fukuoka, southern Japan, in 1945, half a year before the end of
World War II. This was a very challenging time in Japan, and everyone had
difficulty getting basic daily necessities, including food. I myself suffered from
severe malnutrition and was a very sickly child. Around this time my mother
got tuberculosis and spent a long period bed-bound, but she was miraculously
able to recover thanks to a gift of just-developed antibiotics from a family friend
in Hawaii. When I was about 8 years old, my mother was finally able to resume
a normal life. My brother, Kazuo, who is 12 and a half years my senior, left home
to enter the Faculty of Literature at the University of Tokyo as I began elementary school. I was brought up through the efforts of my father and two sisters,
Reiko and Junko, and many other people throughout what was a difficult time
for our family.
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My childhood home was surrounded by nature, with rice paddies, streams,
hills and the sea all nearby. I spent a lot of time outdoors catching fish and picking plants. As an elementary school student, I was engrossed in collecting insects
and watching the night sky. I would say that this intimacy with nature had a
strong influence on me. My brother, every time he returned home from Tokyo
during university holidays, would bring a book, such as carefully chosen works
of George Gamow and Michael Faraday that broadened my awareness of the
world beyond what we learned at school (Faraday, 1861; Gamow, 1940). Being a
weak child, I had no talent for sports and was neither artistic nor gifted in literature. But I managed to achieve good results at school. At the time, I had a vague
longing to be a scientist that was influenced by the expectations of my parents.
University & first experience of research
In high school, I joined the chemistry club, where I first experienced the wonder
of chemical reactions. Partly thanks to this experience, I entered the University
of Tokyo, where I initially hoped to become a chemist, but I had difficulty finding a field to specialise in. Thankfully, this was the time when the central dogma
of molecular biology was just being established. Molecular biology immediately
fascinated me, so I decided to join the lab of Kazutomo Imahori, who was one of
the few scientists running a molecular biology laboratory in Japan at the time.
Imahori’s laboratory had adopted a physicochemical approach to assess interactions between proteins and nucleic acids. Under the direction of Akio Maeda,
I began to study the role of ribosome subunits in protein synthesis. Although I
was unable to produce particularly interesting results, my time in the Imahori lab
was exciting as we participated in the work of a rapidly developing field and I got
my first taste of the joy of experimental work. The experience also provided me
with a strong sense of the continuous process of protein synthesis within cells.
From the second year of my doctoral studies, I decided to move to Kyoto
University, where Maeda had joined the just-founded Department of Biophysics as an associate professor. Kyoto University provided a very free environment,
and I was stimulated by talented new students and friends I met in the biochemistry lab of the Department of Chemistry. Throughout my graduate studies,
the Summer Seminar for Young Biochemists also allowed me to make a large
number of friends from all over Japan who would have a great impact on my
life. In my research, I became interested in colicin E3, a toxic protein that is able
to pass through the membrane of bacterial cells and instantly inhibits protein
synthesis. I think I was also influenced by my growing interest in biological
membranes during this period.
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Post-doc life in New York
After I finished my doctoral studies, I enrolled in the lab of Gerald Edelman at
The Rockefeller University, New York. I was asked was to establish an in vitro
fertilisation system for mouse egg cells, which I reluctantly accepted. Experimentally it was not too difficult, but I had no idea how to examine such fascinating yet few early embryos. After some time, Mike Jazwinski joined our group
from Arthur Kornberg’s lab, where he had been studying phage DNA duplication. Taking inspiration from the elegant study of the cell cycle through the cdc
mutants by Hartwell (Hartwell, 1974), a project to uncover the mechanism of
DNA replication initiation in yeast began in Edelman’s lab, which I joined in
my final year. This was to be my first encounter with yeast as an experimental organism.
We had first planned to use isolated nuclei in this project, but it was difficult
to obtain these organelles in an undamaged state. During our use of densitygradient centrifugation to purify nuclei, I noticed a white layer at the top of
the centrifuge tube. Out of mere curiosity I looked at this white layer under the
microscope, and realised that it was a highly-enriched vacuole fraction. The ease
with which this organelle could be purified left a strong impression on me, and
I think helped to decide the direction of my research.
Return to Japan and work on the vacuole
At the end of 1977 I returned to Japan as an assistant professor in Yasuhiro Anraku’s lab at the University of Tokyo. Anraku’s entire lab was tasked with studying the transporters and the respiratory chain in E. coli. In spite of this, Anraku
allowed me to start a project on yeast, and in retrospect I really appreciate his
decisiveness in affording me this opportunity, which resulted in the beginning
of my nearly 40 years of work in yeast (Figure 1). In those days, the plasma
membrane and its transport mechanisms were the focus of much research. But
I have never been a competitive person, and prefer to work on a subject that few
people are interested in. After a period of reflection, I decided instead to work on
transport across the membrane of an intracellular organelle, the vacuole. While
we now know that elaborate transport systems exist on the vacuolar membrane,
this was by no means a fashionable topic.
At that time the vacuole was thought to be no more than a garbage dump in
the cell, but I thought it must play yet unknown roles in cell physiology. First I
established procedures for purifying vacuoles and making vacuolar membrane
vesicles, and was able to show active transport systems of amino acids and calcium ions over the vacuolar membrane, providing evidence that the vacuole is
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FIGURE 1. Thin-section electron micrograph of a yeast cell. The vacuole is visible as the
large, pale region near the centre of each cell.
important for homeostasis of metabolites and ions (Ohsumi and Anraku, 1981;
Ohsumi and Anraku, 1983). We also described a novel proton-pump on the vacuolar membrane, the V-type ATPase, which generates a proton gradient across
the vacuolar membrane as a driving force of these transport systems (Kakinuma
et al., 1981; Uchida et al., 1985) (Figure 2). These experiences played an important role in the development of my scientific interests, leading into my continued
study of the vacuole and foreshadowing my research on autophagy. Since then,
FIGURE 2. Vacuolar membrane transport systems in yeast. The V-type ATPase maintains
the acidity of the vacuole by pumping H+ into the vacuole, while various active transporters modulate the flow of calcium, amino acids and ions between the cytoplasm and
this organelle.
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the structure and function of the V-type ATPase has become a major field of cell
biology research in its own right.
STARTING MY OWN LAB AT TOKYO UNIVERSITY, KOMABA CAMPUS
In 1988 I was able to start my own lab—consisting of just myself—at the College
of Arts and Sciences at the Komaba campus of the University of Tokyo. My lab
here was modestly equipped, without any fancy instruments. I decided to devote
myself to a related but completely novel task: the lytic function of the yeast vacuole. Since the vacuole is an acidic compartment, due to the function of V-type
ATPase, and contains various hydrolytic enzymes including proteases, I thought
it might be homologous to the lysosome of mammalian cells. However, I had no
well-articulated idea how to test this hypothesis.
Thinking about the life of proteins
Before going into experimental details, I would like to briefly outline protein
metabolism in cells. Proteins, which are a polymer of amino acids, are the key
players in all biological processes. The central dogma of molecular biology states
that protein is synthesised according to the genetic information encoded in DNA
through RNA. As a consequence of this, many researchers throughout the latter
half of the 20th century worked diligently to understand gene expression and its
regulation, and the mechanisms of protein synthesis. Another important consideration that subsequently came to scrutiny was the compartmentalisation of the
cell, as proteins are able only able to properly function after being delivered to the
appropriate part of the cell. This spurred an effort to understand the localisation
of each protein within the cell. Thanks to the efforts of researchers, we now know
that each protein is intricately and precisely trafficked to the correct destination
within the cell according to an intrinsic localisation signal (Figure 3).
However, one aspect that was missing from this picture when I started my
own lab was the fate of proteins, or in other words the question of how intracellular protein degradation occurs. It was known that each protein has a distinct
lifetime, ranging from a few minutes to several months. But for many years, the
degradation of protein was thought to be a passive process that is not particularly
important or interesting.
The significance of protein turnover
I used to begin biology class for first-year undergraduate students with this question: how many red blood cells are made within just one second in your body?
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FIGURE 3. Overview of protein metabolism in cells. In addition to the emphasis on synthesis provided by the central dogma, the delivery, function and eventual degradation of
proteins are important aspects of their life in the cell. The delivery of proteins to various
cellular compartments following translation by the ribosome (purple) is represented at
the bottom of the figure.
The calculation is quite simple and gives an answer of about three million cells
per second. How about hemoglobin in the red blood cell? This number can also
be easily derived, giving about 1x1015—one million billion—hemoglobin molecules per second. It follows that exactly the same amount of cells and proteins
are degraded. The key message is that living things are extremely dynamically
maintained.
The Japanese climate is characterised by four distinct seasons that influence
our culture, and students at school learn that all things are in a cycle of continuous change. Plant leaves offer an excellent example of this concept. In spring,
leaves develop and then actively synthesise starch by sunlight, but in autumn
leaves become red or yellow and fall off (Figure 4). What we see as beautiful
autumn colours is actually caused by the degradation of the leaf ’s photosynthetic machinery: green chloroplasts are completely degraded, and the resulting
amino acids are transported to the trunk, leaving only the red or yellow-coloured
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FIGURE 4. Ginkgo trees throughout the four seasons at the University of Tokyo. The
removal of green chloroplasts in autumn, leaving golden accessory pigments in the leaves,
is one striking example of the importance of degradation in the dynamic cycle of life.
accessory photosynthetic pigments in leaves. Similarly, rice leaves turn yellow at
harvest time. All the protein in the leaves is degraded and transported to make
proteins in rice grains for the next generation. These examples demonstrate that
degradation is not an adverse process, but rather is essential for new construction or regeneration. Let’s think about the human body. Our bodies make about
2–300 grams of protein every day, but we only intake about 70–80 grams of
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FIGURE 5. Protein dynamics in the human body. The bulk of amino acids required for
synthesis are met not through dietary intake, but through the turnover of existing proteins
in the cell.
protein from our diet (Figure 5). The amino acids required for protein synthesis
mostly come from the degradation of the body’s own proteins. Life is maintained
by a tightly controlled balance between synthesis and degradation. Just two years
ago I experienced this personally when I learned that my wife Mariko’s mother
had lived without any food and even water for 13 days. This was another shocking reminder that the recycling of cellular constituents is a potent force in the
robust nature of life.
Historical perspectives on protein turnover
The establishment of the central role of proteins in biology was a very important
development in modern biological science. In contrast to the process of synthesis
emphasised by the central dogma, when I began my research the degradation
of proteins was seen as a passive process that was not particularly important by
many of my contemporaries. In this regard Rudolph Schoenheimer was a pioneer
in the field, as he had proposed the concept of “protein turnover” in the 1930s
following his development of isotopes to follow metabolic processes (Schoenheimer et al., 1939; Schoenheimer, 1942) (Figure 6A). Unfortunately, however,
his contention was controversial and not widely accepted. After this, Christian
de Duve, recipient of the Nobel Prize in 1974, used cell fractionation to discover
the lysosome, an organelle containing a range of degradative enzymes (De Duve
Molecular Mechanisms of Autophagy in Yeast
(a)
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(b)
FIGURE 6. (a) Rudolph Schoenheimer and (b) Christian de Duve.
et al., 1955) (Figure 6B). Researchers at The Rockefeller University subsequently
observed the delivery of extracellular material to the lysosome using electron
microscopy (Novikoff et al., 1956). The delivery of intracellular material including organelles such as mitochondria to the lysosome was also observed. De Duve
referred to these processes as ‘heterophagy’ (now known as endocytosis) and
‘autophagy’, respectively (de Duve, 1963). Autophagy, from the Greek for ‘selfeating,’ was described by The Rockefeller University researchers as having several
hallmarks. Firstly, a membrane sac appears and enwraps a portion of the cytoplasm, forming a double-membrane structure, the autophagosome. Next, the
outer membrane of the autophagosome fuses with the lysosome, and finally the
inner membrane and its contents are degraded within the lysosome.
Autophagy research was continued by two key groups: one, led by Glenn
Mortimore (Figure 7A), conducted detailed physiological and biochemical analyses of the phenomenon using liver perfusion in rats (Mortimore and Ward,
1976; Mortimore et al., 1983), while a second group, led by Per Seglen, examined
the effect of nutrient starvation on autophagy and the regulation of autophagy,
mainly using cultured cells (Seglen and Gordon, 1982; Seglen et al., 1980) (Figure
7B). However, as electron microscopy was the only means of assessing autophagy
at the time and the lysosome is a dynamic organelle, most works focused on the
analysis of membrane dynamics in autophagy, leaving the genes and proteins
implicated in the autophagy mechanism unidentified for many years. A simpler
model system was required to make further progress in the field.
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(b)
FIGURE 7. (a) Glenn Mortimore and (b) Per Seglen.
In the meantime, another intracellular degradation system, the ubiquitin/
proteasome system, was discovered. As it became clear that this system plays
important roles in the regulation of a variety of cellular processes, protein degradation suddenly attracted a huge amount of attention in biological research. This
was ultimately recognized by the Nobel Prize in Chemistry in 2004, which was
awarded to Aaron Ciechanover, Avram Hershko and Irwin Rose, but the field
of lysosomal degradation and autophagy remained quiet. Now it is thought that
these two systems function together as the major means of cytoplasmic protein
degradation.
Discovering autophagy in yeast by light microscopy
Assuming that the vacuole functions as a lytic compartment, the questions I
had were when, what and how cytoplasmic constituents go across the vacuolar
membrane and become accessible to vacuolar enzymes. First, I searched for a
condition where massive protein degradation might occur in the yeast life cycle.
Spore formation is a meiotic process that generates four spores and is induced by
the depletion of nitrogen, the essential element of amino acids (Figure 8). I reasoned that this dramatic cellular remodelling should require the degradation of
pre-existing proteins to make the proteins necessary for this cell differentiation.
I have to admit that I’ve always loved to observe yeast cells by light microscopy. The vacuole is relatively large, is the only clearly visible organelle in the cell
and contains a salt-like solution that has a very low concentration of protein, so I
knew that it would be easy to see structures if they were inside. I probably spent
more time sitting at the light microscope than anyone else during my studies of
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FIGURE 8. Morphological changes associated with sporulation in yeast. The onset of nitrogen starvation causes diploid cells to undergo meiosis and cell division, forming four
spores in a process that requires vast remodelling of the protein component of the cell.
N = nucleus, V = vacuole, CW = cell wall, M = mitochondrion.
the vacuole, looking for morphological changes that might provide hints about
the function of this dynamic organelle.
I carefully observed the early stages of the sporulation process, but couldn’t
see any obvious changes in the vacuole. I thought that this may be due to the
degradation of structures in the vacuole. I conceived that if I were to use a cell
lacking vacuolar proteases, structures delivered to the vacuole for degradation
would remain in this organelle and might be visible. Fortunately, the well-known
yeast geneticist Elizabeth Jones had donated many proteinase mutants to the
Yeast Genetic Stock Center at UC Berkeley, so I obtained these strains and shifted
to nitrogen depleted medium.
Looking down the microscope, I saw many spherical structures vigorously
moving around inside the vacuole. These structures appeared within 30 minutes and almost completely filled the vacuole after 3–4 hours (Figure 9). For
me, this observation marked the exact starting point of more than 27 years of
autophagy research. The reason that I was able to discover this phenomenon
with only a common low magnification light microscope owes to the vigorous
movement of these structures by Brownian motion. I was also fortunate that the
structures, which we now call autophagic bodies, were large enough to be visible under a light microscope. In the otherwise tranquil yeast cell, this dramatic
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FIGURE 9. Accumulation of autophagic bodies within the vacuole under nitrogen starvation, as observed by bright-field (top) and phase contrast (bottom) light microscopy.
phenotypic change really struck me, and I remember staring down the microscope for some hours, unable to tear my gaze away. I knew that I had encountered
an unknown and fascinating phenomenon; this was the starting point of my
autophagy research.
Electron microscopy and the morphology of autophagy
I next called on the excellent electron microscopists Misuzu Baba and Masako
Osumi to capture the morphology of this phenomenon by electron microscopy.
Their efforts produced beautiful images that provided irrefutable evidence of
the topological features of the entire autophagy process (Baba et al., 1994), as
shown in Figure 10.
A thin section image of a nitrogen starved cell is shown in Figure 10A. Spherical structures can be seen within vacuole, and the high-magnification image
presented in Figure 10B shows that these structures are bound by a single membrane. We can see that their contents are exactly the same as the cytoplasm, containing the same density of ribosomes and various cytoplasmic structures, such
as the rough ER observed in Figure 10C, suggesting nonselective sequestration
of cytoplasmic components. A membrane sac next to the vacuole, the isolation
membrane, is also visible in Figure 10C enwrapping a portion of the cytoplasm.
After completion of membrane expansion and sealing, a double membrane
structure, the autophagosome, is formed (Figure 10D). Freeze-fracture imaging (Figure 10E) shows the fusion event of an autophagosome with the vacuolar membrane, where the outer membrane of the autophagosome is continuous with the vacuolar membrane (Baba et al., 1995), and the inner membrane
structures that we named autophagic bodies are also present. Autophagic bodies
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FIGURE 10. Electron micrographs showing topological phenomena associated with nitrogen starvation-induced autophagy. (A) Autophagic bodies within the vacuole, (B) highmagnification image of autophagic bodies, (C) cup-shaped isolation membrane adjacent
to the vacuole, (D) an autophagosome adjacent to the vacuole, (E) freeze-fracture image
of an autophagosome fusing with the vacuole, (F) mitochondria within autophagic bodies
delivered to the vacuole.
occasionally contain mitochondria, as can be seen in Figure 10F, indicating that
autophagy can degrade not only cytosolic protein but also supramolecular structures, such as ribosomes and even entire organelles, which is a characteristic
feature of autophagy. This is in contrast to the specific recognition of smaller,
individual targets by the ubiquitin/protease system. Autophagosomes are transient, ephemeral structures, and autophagic bodies are degraded immediately
in the vacuole in wild-type cells, features that prevented their identification for
many years.
A scheme of the autophagic process in yeast is presented in Figure 11. When
cells are faced with starvation conditions, a small membrane sac appears next to
the vacuole and expands to enwrap a portion of cytoplasm. Then, a double membrane structure, the autophagosome, targets the vacuole and fuses with the vacuolar membrane, releasing the inner membrane structure into the vacuole. In wild
type cells these structures are immediately disrupted by vacuolar enzymes, and
their contents are degraded for reuse. The proteinase-deficient mutant allowed us
to follow the progression of autophagy as the accumulation of autophagic bodies.
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FIGURE 11. Schematic diagram of nitrogen starvation-induced autophagy in yeast.
Importantly, while the vacuole is a vastly larger organelle than the mammalian lysosome, the entire process that we observed was topologically the same
as macroautophagy in mammalian cells, confirming that yeast could be used
as a model organism in the study of autophagy (Baba et al., 1994). Kazuhiko
Takeshige was also able to show early on that this phenomenon is not restricted
to sporulation, but is a general cellular response to a range of nutrient starvation
conditions (Takeshige et al., 1992). These factors gave the first indication that
autophagy is a general process that is induced in diverse organisms in response
to a range of stimuli, further convincing me that we had discovered an important
cellular process.
Identifying autophagy-related genes in yeast
Yeast is a very good organism for the dissection of complicated biological phenomena through genetic analyses. In this regard I was inspired by the beautiful
works of Lee Hartwell, whose group uncovered details of the cell cycle through
their analysis of the cdc mutants (Johnston et al., 1977), and also by the intricate
analysis of the secretory pathway using sec mutants by Randy Schekman’s group
(Novick et al., 1980). These contributions were recognized with the Nobel Prize
in Physiology or Medicine in 2001 and 2013, respectively.
As we knew nothing of the genes and proteins that are involved in the autophagy mechanism, I thought that the isolation of mutants defective in autophagy
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was the first step in opening up the field of autophagy. However, we had no
idea what an autophagy-defective phenotype would look like. At this point, we
decided to use autophagic body accumulation in the vacuole as a morphological indicator of autophagy progression. My first Master’s course student, Miki
Tsukada, was central at this stage of our work and did an excellent job isolating
autophagy defective mutants. We took mutagenised cells, individually subjected
them to starvation and checked for the accumulation of autophagic bodies,
aiming to isolate cells in which autophagic bodies were not observed. Using this
approach, we identified the first autophagy mutant strain, which we called apg1,
now referred to as atg1 (Tsukada and Ohsumi, 1993). We then confirmed, by
the failure to induce protein degradation in this strain, that the autophagic body
is indeed an intermediate structure of the degradation process. We also found
that, as expected, the homozygous diploid mutant was not able to sporulate.
However, under standard growth conditions in the nutrient rich medium YEPD,
no difference was observed between the atg1 strain and wild-type cells, and there
appeared to be no obvious defect in vacuolar function or secretion.
It seemed impossible that such an intricate pathway of delivery to the vacuole could be controlled by a single gene, and I was convinced that we should be
able to identify many more. We found that atg1 cells that were subjected to prolonged nitrogen starvation lose viability faster than their wild-type counterparts.
Assuming this loss of viability phenotype was caused by the defect in autophagy,
we next conducted a primary screen looking at the viability of mutant strains. By
isolating strains in which viability was reduced and then performing a secondary
screen examining these strains for vacuolar morphology defects, we were able to
identify roughly 100 autophagy-defective strains at once (Tsukada and Ohsumi,
1993) (Figure 12). Further genetic analysis showed that 14 complementation
groups were among these mutants. As we now know that in nitrogen starved
cells 18 genes are essential for autophagy, I can say that this refined approach
was extremely effective. We initially predicted that these mutant strains would
be characterised by defects at various stages of the autophagy mechanism, but as
it turned out all of these genes were essential for the formation of the autophagosome, the most important process in autophagy.
This was a morphological screen, based on the inability to accumulate
autophagic bodies in the vacuole. We therefore eliminated mutants characterised by a severe growth defect, cells lacking essential genes and also strains with
abnormal vacuolar morphology. In addition, as we used the decline in viability
as a primary screen, which only appears with a complete block of autophagy
function, we only identified autophagy null mutants, to the exclusion of partially
defective mutants.
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FIGURE 12. Overview of the experimental approach used to isolate autophagy-defective
mutants. This screen allowed us to identify most of the ATG genes in a short period of time.
Next, we set out to clone and identify the genes implicated in the mutant
phenotypes observed in the screen. Tsukada was the first to succeed, cloning the
ATG1 gene. Through the sequencing of ATG1, we learned that this gene encodes
a protein kinase (Matsuura et al., 1997). However, as we identified the remaining genes, starting with Kametaka’s work on ATG5 (Kametaka et al., 1996) and
ATG6, we found that all the other ATG genes encoded unidentified proteins that
were otherwise not essential for growth in nutrient replete conditions and had
no phenotypic implications for cells other than a defect in autophagy. Therefore, although we had identified nearly all the genes responsible for autophagy,
we were unable to deduce anything about their function from their amino acid
sequences and went through a difficult period where we were unable to officially
announce our findings.
Yeast had been the subject of comprehensive genetic analysis throughout
the world for many years at this point, and it was very surprising that autophagy
genes had somehow escaped the attention of other researchers. I suspect that this
is because researchers were interested in essential genes, and the conventional
definition of an essential gene was the cell’s inability to grow in nutrient rich
medium when the gene in question was disrupted.
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One important product of our time at the College of Arts and Sciences of
Tokyo University that deserves mention was Takeshi Noda’s establishment of an
assay of autophagic activity (Noda and Ohsumi, 1998; Noda and Klionsky, 2008).
Noda constructed a strain in which the precursor of the vacuolar alkaline phosphatase Pho8 lacks its N-terminal vacuolar targeting sequence, and expressed
this precursor in the cytosol. Upon the induction of autophagy, a portion of the
modified version of Pho8 would be taken up by the non-selective activity of
autophagy and delivered to the vacuole. Here, the precursor is processed to yield
the mature, enzymatically active form of Pho8. Therefore, we were able to quantitatively measure the extent of autophagy by assessing Pho8 activity. This assay,
in which degradation can be quantitatively detected through the appearance of
enzymatic activity, has been an excellent and widely-used tool in the analysis of
autophagic activity that is still used today. I hope that this type of enzymatic assay
can also be developed for use in other organisms, especially mammalian systems.
In this way, my career in autophagy research began in that small laboratory at the Komaba campus. The discovery of autophagy by light microscopy, its
morphological analysis by electron microscopy, the isolation of mutant strains
implicated in autophagy and the beginning of the identification of genes responsible for the process were all achieved within eight years.
A GOLDEN AGE OF AUTOPHAGY RESEARCH AT NIBB
In 1996, I became a professor at the National Institute for Basic Biology (NIBB)
in Okazaki, which provided us with a very conducive research environment. I
asked Tamotsu Yoshimori to join our lab as an associate professor to start work
on autophagy in mammalian cells, and also Takeshi Noda and Yoshiaki Kamada
to join us as assistant professors. The following year, Noboru Mizushima came
to my lab as a postdoctoral researcher, and later as an assistant professor. Year by
year, talented postdoctoral researchers and graduate students gathered to my lab.
In 1997, just after we started at the NIBB, we also organised the first International
Symposium on Autophagy (ISA) meeting, which we held at the institute. This
forum for the discussion of autophagy research has proven to be very successful,
and we will hold the 8th ISA in 2017, the largest yet.
Thanks to the NIBB, we were able to produce a truly unique research environment where our work in yeast was accompanied by studies in mammalian
and plant cells. I was expecting that it would take a long time to identify all the
genes required for autophagy, but thanks to our collaboration with Mariko’s laboratory at the Teikyo University of Science, which helped with cloning, as well as
the sequencing of the entire yeast genome in 1996, we were able to elucidate the
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FIGURE 13. Immunoblot analysis of the Atg12 protein. (Left) The appearance of a band
of much higher molecular weight than expected for Atg12 is indicated by an asterisk.
(Right) The high molecular weight band disappears in cells lacking ATG5, ATG7 or
ATG10, but not ATG1.
genetic context of nearly all the ATG genes in a relatively short period. With our
subsequent effort to identify interacting proteins of the characterised ATG gene
products, we were able to confidently identify all 18 of the genes essential for
autophagy under nitrogen starvation conditions. From this point, we focused on
the characterisation of the Atg proteins and the mechanistic details of autophagy.
The Atg12 conjugation system
The first breakthrough was made by Mizushima when he examined the only
just cloned Atg12 protein (Mizushima et al., 1998a). Using immunoblotting,
he noticed two bands corresponding to Atg12, one of which indicated a much
greater apparent molecular weight than that predicted from the protein sequence
(Figure 13). In the absence of Atg5, Atg7 or Atg10, however, the high molecular
weight band disappeared. This was the first indication we found of a relationship
between Atg proteins, and within a short period we were able to demonstrate
that these proteins together constituted a unique ubiquitin-like conjugation
pathway. Although Atg12 is a vastly larger protein than ubiquitin, it ends with
a glycine residue at its C-terminus and is expressed as a mature protein without
any additional residues. Atg12 is then activated by Atg7, an E1 enzyme, to yield a
thioester intermediate that is then transferred to the E2 enzyme Atg10, before it
ultimately forms an isopeptide bond at a lysine residue at the centre of the Atg5
molecule. This Atg12-Atg5 conjugate then binds to a dimeric form of Atg16,
yielding a functional dimer of Atg12-Atg5—Atg16 molecules (Figure 14A). A
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FIGURE 14. The six functional groups of Atg proteins: (A) the Atg12-Atg5 conjugation
system, (B) the Atg8-PE conjugation system, (C) the Atg1 protein kinase complex, (D)
the autophagy-specific PI3 kinase complex I, (E) Atg9 vesicles and (F) the Atg2-Atg18
complex. The concerted action of all groups is necessary for autophagosome formation.
unique feature of this conjugation reaction is that Atg5 is the sole target of the
system and the reaction is not regulated by environmental conditions, such as
starvation. With the elucidation of this conjugation system, we were able to characterise the functions of five Atg proteins in one series of experiments. However,
why such an elaborate conjugation system is required to form this functional
complex remains a mystery.
The Atg8 conjugation system
We were also interested in the small protein Atg8. We knew from immunoelectron microscopy that Atg8 localises to the isolation membrane and autophagosome, and were therefore hopeful that this protein could be used as a marker of
membrane formation in autophagy (Figure 15). During these studies, the work of
two graduate students, Takayoshi Kirisako and Yoshinobu Ichimura, unveiled a
second ubiquitin-like conjugation system (Ichimura et al., 2000). Atg8 is another
ubiquitin-like protein that is involved in autophagy that contains an arginine
residue at its C-terminus. This arginine is cleaved by the cysteine protease Atg4
to expose a glycine residue, giving the mature form of the protein (Kirisako et
al., 2000). The mature Atg8 molecule is then activated and transferred to Atg3
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FIGURE 15. Immunoelectron micrograph showing Atg8 (dark spots) residing on the
membrane of an autophagosome.
by Atg7, which is the same E1 enzyme as found in the Atg12 conjugation system.
However, we weren’t able to detect the final product of this reaction by conventional SDS-PAGE analysis. We soon found that most Atg8 resides as a tightly
membrane-bound form, and when we purified this bound form we determined
that the apparent molecular weight had increased by approximately 635 daltons. Through our collaboration with Toshifumi Takao of Osaka University, we
were also able to attribute the binding of Atg8 to a membrane phospholipid,
phosphatidylethanolamine, which we confirmed by isolating Atg8-PE using urea
SDS-PAGE (Figure 14B). This attracted a lot of attention because the target of
conjugation was not a protein but a lipid, which is very unusual for this type of
reaction. We were also able to reconstitute the Atg8 conjugation system in vitro
(Ichimura et al., 2004), which eventually allowed us to show that Atg8 is involved
in membrane tethering and hemifusion that supports the expansion of the isolation membrane (Nakatogawa et al., 2007).
Thus, to our surprise, we found that about half of the ATG genes are involved
in these two conjugation systems. Both Atg12 and Atg8 are activated by the
same E1 enzyme, Atg7, before the protein is transferred to distinct E2 enzymes,
Atg10 or Atg3, respectively. In addition, we also found that the Atg12 system
is required for the conjugation of Atg8 to PE, further hinting at the close relationship between these two systems (Hanada et al., 2007). Recently, it has been
demonstrated that the Atg12-Atg5 conjugate induces a conformational change in
Atg3, promoting Atg8-PE formation (Sakoh-Nakatogawa et al., 2013). Structural
details of the complexes involved in these reactions were solved thanks to our
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FIGURE 16. Schematic overview of the two ubiquitin-like conjugation systems involved in
autophagosome formation. Structures of the associated proteins are included in the figure.
close collaboration with Fuyuhiko Inagaki and Nobuo Noda’s groups, but details
of their functions in the cell remain to be uncovered (Figure 16).
Events during autophagy induction and their regulation
In 1998, Takeshi Noda found that rapamycin induces autophagy even when cells
are grown in rich medium (Noda and Ohsumi, 1998). As rapamycin is an inhibitor of TOR kinase, which is an important nutrient sensing complex, we therefore
proposed that TOR kinase is the most upstream regulator of autophagy. Meanwhile, work by our group led by Tomoko Funakoshi and Yoshiaki Kamada also
discovered the important role of Atg13 in the activation of Atg1 kinase function
(Kamada et al., 2000). Atg13 is one of the targets of TOR kinase and functions
as the key regulator of autophagy induction (Figure 14C). When nutrients are
replete in the environment, Tor kinase heavily phosphorylates Atg13, blocking
its interaction with Atg1. Upon starvation, Atg13 is quickly dephosphorylated,
however, and associates with Atg1, subsequently facilitating its interaction with
other Atg proteins. This crucial upstream event is essential for the induction
of autophagy. Kamada also led the discovery of Atg17 (Kamada et al., 2000),
which interacts with Atg13. Subsequently two other proteins, Atg29 and Atg31,
were found by Tomoko Kawamata and Yukiko Kabeya (Kawamata et al., 2005;
Kabeya et al., 2007). These latter two proteins form a stable complex with Atg17,
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and together with Atg1 and Atg13 this complex, known as the Atg1 complex,
is essential for the induction of autophagy under conditions of starvation, as
described below.
The autophagy specific PI3 kinase complex I
Although every other ATG gene encoded novel proteins that had not been
described elsewhere, ATG6 turned out to be the same gene as VPS30, which
had only just been discovered by Scott Emr’s group as a protein of the vacuolar
protein sorting (Vps) pathway (Seaman et al., 1997). This attracted our attention,
and through biochemical analyses led by Akio Kihara we discovered that this
protein is actually a component of a PI3 kinase complex essential for autophagy,
comprising Vps34, Vps15, Atg14 and Vps30, which we termed PI3 kinase complex I (Kihara et al., 2001) (Figure 14D). Interestingly, PI3 kinase complex II,
which is involved in the Vps pathway, contains Vps38 in the place of Atg14,
and these subunits are responsible for the appropriate localisation of the complexes to play their specific function. Following this, Yasuhiro Araki uncovered
yet another specific component of PI3 kinase complex I, Atg38 (Araki et al.,
2013). This protein localises to the isolation membrane and is essential for the
incorporation of the product of PI3 kinase, PI3P, into the isolation membrane,
with which it is ultimately delivered to the vacuole
Atg9 and the supply of membranes to the isolation membrane
An unsolved mystery in the field is the origin of membranes that are supplied to
the autophagosome during its formation. A protein that attracted much attention for its role in membrane expansion is Atg9, the only multiple-pass transmembrane protein among the Atg proteins (Figure 14E). There has been much
speculation about the localisation of Atg9 within the cell, but we observed that
Atg9 resides mostly on small membrane vesicles arising from the Golgi apparatus
that freely move throughout the cytoplasm (Yamamoto et al., 2012). Our analyses
indicated that a small number of Atg9 vesicles are required for the early stages
of the formation of a single autophagosome, suggesting that these vesicles form
seed membranes that incorporate membranes from other sources for isolation
membrane expansion.
Atg2 and Atg18
In addition, Atg18, which was first identified by Michael Thumm’s group, contains
many WD repeats and binds with PI3P to form a complex with Atg2, thereby
exerting its essential function in both the Cvt pathway and autophagy (Barth et
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al., 2001) (Figure 14F). Atg2, on the other hand, is very large protein that while
essential for autophagy does not contain any known functional domains, and its
role in the mechanism of autophagy remains poorly understood. Considering all
these findings, we therefore concluded that a total of 18 Atg proteins are essential
for autophagy, and that these proteins can be classified into the six functional
groups presented in Figure 14: the Atg1 kinase and its regulators, the specific PI3
kinase complex, two conjugation systems, the sole membrane spanning membrane protein Atg9, and the Atg2-Atg18 complex.
FIGURE 17. Atg proteins, tagged with GFP, at least partially localise to the PAS, indicated
by RFP-tagged Ape1. In response to an induction signal, Atg proteins, usually moving
freely throughout the cytosol, associate with the PAS near the surface of the vacuole,
facilitating the progression of autophagy.
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Identification of the PAS
Although we had identified the core machinery of autophagosome formation,
the next questions were where and how these proteins function in the cell.
Kuninori Suzuki, who was observing Atg8 localisation under starvation conditions, noticed that these proteins form a dot near the vacuole from which the
autophagosome subsequently generated (Suzuki et al., 2001). We called this dot
the Pre-Autophagosomal Structure (PAS). By fusing each Atg protein with GFP
and confirming their continued function, Suzuki found that all Atg proteins
at least partially localise to the PAS under starvation conditions (Suzuki et al.,
2007) (Figure 17). We next devised a systematic approach to understand the
relationship between each Atg protein and the PAS by assessing the localization
of Atg proteins in strains lacking individual ATG genes. Through this work, we
learned that the six functional groups of Atg proteins associate with the PAS in
a hierarchical manner (Suzuki et al., 2007) (Figure 18). Atg17 exists at the top of
FIGURE 18. Overview of the sequential nature of Atg protein recruitment to the PAS.
Upon starvation, a scaffold complex is formed through the interaction of dephosphorylated Atg13 with Atg1 and the Atg17-Atg29-Atg31 complex at the vacuolar membrane (I).
Membrane recruitment then begins as Atg9 vesicles associate with the PAS (II) before PI3
kinase complex I joins the PAS to contribute PI3P to the expanding isolation membrane
(III). Atg2 and Atg18 then interact with the PAS through an unknown mechanism that
is essential for autophagy progression (IV). Finally, Atg8-PE, a product of the Atg12 and
Atg8 conjugation reactions, plays a role in membrane expansion.
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this hierarchy, and the association of other Atg proteins follows a strict order of
precedence. This study was at the forefront of the many molecular studies of the
PAS that followed, becoming a field in its own right.
Thanks to these fluorescence microscopy studies of the spatiotemporal
dynamics of Atg proteins, we now know that most Atg proteins exist in a diffuse localisation during nutrient replete conditions, free to move randomly
through the cytosol. The onset of starvation causes a fraction of these proteins
to be recruited to the PAS. The hierarchical relationship amongst Atg proteins
describes the order and timing of each Atg protein’s recruitment to the PAS.
Mammalian ATG genes and the expansion of autophagy research
Also during our time at NIBB, Mizushima and Yoshimori began to study mammalian autophagy, and successfully showed that the Atg12 conjugation system is
well conserved in mammalian cells (Mizushima et al., 1998b). Yoshimori definitively proved that LC3, the yeast Atg8 homologue, can be used as a very effective
marker of autophagy progression (Kabeya et al., 2000). At around this time, two
of my colleagues, Hideki Hanaoka and Kohki Yoshimoto, succeeded in identifying ATG genes in plant cells (Yoshimoto et al., 2004). Although the homology
of ATG genes is low, the ATG system is well conserved between yeast, mammals
FIGURE 19. Expression of GFP-LC3 in tissues of a transgenic mouse. Shown are the localisation of autophagosomes, observed as puncta, in skeletal muscle (A, from a starved adult
mouse), the heart (B, from a neonate), an embryo (C, post-fertilisation) and the pancreas
(D, from a mouse with acute pancreatitis), as observed by fluorescence microscopy.
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and plants, indicating that the core machinery of autophagy was acquired at an
early stage of evolution.
The identification of ATG genes essential for autophagy changed the entire
landscape of autophagy research. One aspect of this revolution is that we can now
visualize the process of autophagy by fluorescence microscopy using Atg proteins
as markers. Mizushima took advantage of this when he constructed a transgenic
mouse expressing GFP-LC3 (Mizushima et al., 2004) (Figure 19). This mouse has
allowed us to assess the degree to which autophagy occurs in every organ of the
mouse’s body and has been shared with labs all over the world. Mizushima also
developed the first ATG gene knockout mouse, which allowed us to show that
autophagy plays a crucial role for survival at birth (Kuma et al., 2004). Masaaki
Komatsu was also able to demonstrate that conditional knockout of the ATG7
gene causes accumulation of ubiquitinated proteins and ultimately tumour in
the liver (Takamura et al., 2011). From this point, manipulation of ATG genes
in various organisms, different cell types, a variety of tissues and organs, and
individuals started in many labs around the world and revealed a vast array of
physiological functions of autophagy (Figure 20). The relevance of autophagy
in various diseases is becoming clear. This rapid expansion of autophagy would
not have been possible without the concerted efforts of autophagy researchers
all over the world. But this expanding area falls outside the scope of this lecture,
which focuses on my fundamental research in yeast.
FIGURE 20. The diverse physiological roles of autophagy in mammalian cells.
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STARTING MY FINAL LAB AT THE TOKYO INSTITUTE OF TECHNOLOGY
In 2009, another important change occurred in my career. I was offered a position as a specially appointed professor at the Tokyo Institute of Technology,
where we were given access to excellent research facilities. I brought along many
colleagues from the NIBB to this institute, and with Hitoshi Nakatogawa and
Kuninori Suzuki as assistant professors initially, later joined by Assistant Professors Hayashi Yamamoto, Yasuhiro Araki and Tomoko Kawamata, we started a
new laboratory. Here, we have been conducting increasingly advanced analyses of the Atg machinery and more recently have been considering the role of
autophagy as one of many cellular processes.
Atg13 and its role in Atg protein assembly at the PAS
Yamamoto and Nobuo Noda worked together to uncover details of the early
steps of PAS organisation. Atg13, a key player, has a unique structure that is
made up of an N-terminal globular domain and a long, naturally disordered
C-terminal region. This disordered region contains many amino acid residues
that are phosphorylated by Tor1 kinase. As mentioned above, Atg13 is quickly
dephosphorylated upon starvation, which allows it to bind to Atg1 and Atg17 via
specific sites in the disordered region. Atg17 exists as a dimer of Atg17-Atg29Atg31 complexes, the structure of which was recently solved (Ragusa et al., 2012).
As a result, a dimeric complex of Atg1, Atg13, Atg17, Atg29 and Atg31 is formed,
known as the Atg1 complex. However, work by Dan Klionsky’s group had suggested that the actual number of Atg proteins assembling at the PAS is actually
30–40 (Geng et al., 2008). It was therefore clear that further assembly was occurring. Recently, we found that Atg13 has a second, weak Atg17 binding site that is
also essential for PAS formation and autophagy (Yamamoto et al., 2016). Atg13 is
not able to use both of the binding sites to associate with a single Atg17 protein,
but rather links two Atg17 molecules, resulting in the formation of the PAS scaffold. In this way, the scaffold can form a large assembly (Figure 21).
Why is such a higher-order supramolecular structure necessary for autophagy? The answer lies in the activation of Atg1 kinase. Within the PAS scaffold,
this higher-order organisation brings Atg1 kinases into proximity of each other,
which results in auto-phosphorylation between adjacent Atg1 molecules (Figure
21). This activation of Atg1 kinase activity facilitates the next step of membrane
formation, the recruitment of Atg9 vesicles to the PAS, contributing the initial
membrane for isolation membrane expansion. Recently, Sho Suzuki found that
the N-terminal HORMA domain of Atg13 binds to the N-terminal disordered
region of Atg9 (Figure 22) (Suzuki et al., 2015). Assembly of many HORMA
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FIGURE 21. Formation of the supramolecular Atg1 complex by higher-order interactions
of Atg13 molecules. Dephosphorylation of Atg13, causing Atg1 complex formation, is
followed by inter-complex linkages that facilitate the activation of Atg1 kinase. Activation is achieved by autophosphorylation by adjacent Atg1 proteins enmeshed within the
surpramolecular complex.
domains might be essential for interaction with Atg9 on the recruited vesicles.
These findings together demonstrate that the PAS is not static, but is in fact a
dynamic and flexible structure consisting of a large number of Atg proteins and
membrane structures. During membrane formation, the PAS must be regulated
spatiotemporally in a concerted manner by protein modification and the formation of a transient assembly, but we still need to extend our analyses to the later
steps of autophagosome formation to get a clear picture of these events.
Alternate forms of autophagy
I mentioned earlier that nutrient starvation is the most potent signal for the
induction of autophagy, representing a fundamental evolutionary adaptation to
the persistent challenge of nutrient limitation, starvation. In our work, we have
predominantly assessed autophagy in cells that have been subjected to starvation. But evolution is highly resourceful, and as with many highly-conserved core
cellular processes, autophagy has found a range of diverse applications in the
cell. In contrast to the canonical autophagy pathway, where induction is tightly
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FIGURE 22. 3D model of the interaction between the supramolecular Atg1 complex
(center) and Atg9 vesicles (membranes in pink, Atg9 in red). This interaction supports
the delivery of the first membranes to the PAS, and is essential as the seed of the isolation membrane.
regulated and induced in response to starvation, constitutive forms of autophagy,
which occur continuously at a basal level in the cell, have also been uncovered.
Constitutive autophagy, the progress of which is independent of starvation,
is important for the maintenance of cytoplasmic homeostasis. Dan Klionsky
was central in the uncovering of one such form of constitutive autophagy, the
Cvt pathway, which is an anabolic process that depends on the core autophagy
machinery (Umekawa and Klionsky, 2012). In the Cvt pathway, the receptor protein Atg19 allows Ape1 complexes to interact with the PAS. Following this, the
core autophagic machinery then very selectively packages the Ape1 complexes
into a Cvt vesicle. This vesicle is then delivered to the vacuole in the same manner
as the much larger autophagosome. Once delivered, the cargo of peptidases is
released into the vacuolar lumen, where they are then processed to yield the
mature, active hydrolases. This demonstrates that evolution has resulted both
in selective applications of the autophagic machinery and anabolic processes in
the cell, aside from its widespread induction in response to cellular crises such
as nutrient deprivation.
This is not the only form of selective autophagy, which is now a burgeoning
field in autophagy research. The targeted isolation and degradation of a range
of cellular components, including specific proteins, supramolecular complexes,
protein aggregates, organelles and even viruses and invasive bacteria (Amano
et al., 2006) (Figure 23). As an example, Koji Okamoto, in our lab, identified
Atg32, a receptor protein for the selective degradation of mitochondria that was
concurrently identified by Klionsky’s group (Okamoto et al., 2009; Kanki et al.,
2009). Our analysis showed that Atg32 interacts with Atg11, adaptor protein for
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FIGURE 23. Selective autophagy in yeast. In contrast to starvation-induced autophagy,
selective autophagy allows the degradation of specific cargo using the core autophagy
mechanism. Proteins involved in the selective degradation of peroxisome (purple),
mitochondria (blue), the ER (green) and the maturation of Ape1 (teal) are shown in the
coloured boxes. Receptor proteins are indicated in red.
selective autophagy, which allows the canonical autophagy mechanism to isolate
the targeted organelle for vacuolar degradation. In yeast, receptor proteins for
the peroxisome (Atg28, Stasyk et al., 2006) and α-mannosidase (Atg34, Suzuki
et al., 2010) have been uncovered in addition to Atg32, and Nakatogawa’s group
recently discovered receptors for selective autophagy of the ER and the nucleus
(Atg39 and Atg40, Mochida et al., 2015). These receptor proteins, of which many
more will be identified, carry an Atg8-interacting motif, as well as an Atg11
binding site. Atg11 is essential for all forms of selective autophagy, serving as a
scaffold much like the Atg17 complex involved in starvation-induced autophagy.
The mechanism by which individual organelles are marked for degradation is
not clear and further research is essential, but it appears that selective autophagy
plays a role in maintaining the efficient function and health of intracellular components, removing organelles as they age or accumulate damage. Autophagy
therefore also plays a role in the maintenance of cellular homeostasis, regardless
of environmental circumstances such as starvation.
Current work
Autophagy research is still at a very early stage, and our understanding of the
physiological role of autophagy is only in its infancy. Over twenty years, our
Molecular Mechanisms of Autophagy in Yeast
307
functional analysis of the Atg proteins allowed us to focus on one question: how
the autophagosome is formed. To this end, we predominantly used cells that
were shifted from nutrient rich conditions to a synthetic medium lacking nitrogen. This is because the induction of bulk autophagy is most striking using this
experimental regime, and is thereby easiest to analyse. However, such nutrient
rich conditions very rarely exist in nature, and I believe that we must endeavour
to understand the contribution that autophagy makes during the natural life
of yeast cells. From this perspective, I feel that we have returned to the starting
point of autophagy research by going back to the original, fundamental questions—when, how, and what is degraded by autophagy? Yeast still offers striking
advantages in comparison to mammalian cells, including the ease of vacuole
purification and biochemical analyses. However, a major weakness in the study
of autophagy has been the relatively limited repertoire of biochemical techniques
available to researchers. Whilst developing new techniques, we must systematically address the conditions that induce autophagy, identify specific degradation
targets, determine how degradation occurs within the vacuole and identify the
final products of degradation and how they are exported back to the cytoplasm.
We must extend our consideration of degradation to targets other than protein,
which has been the focus until now, to include other cellular material, such as
nucleic acids, lipids and polysaccharides. As an example, our group has recently
shown bulk RNA degradation by autophagy, and that the nucleotides resulting from this process are secreted to the outside of the cell (Huang et al., 2015)
(Figure 24). In order to capture such fascinating phenomena, the identification
of the products that arise from autophagic degradation, the mechanism of their
export from the vacuole as well as their effect on and implications for cellular
metabolism have to be considered. I believe that this research will deepen our
understanding of degradation and also help to characterise the ambiguous relationship between genetics, phenotype and disease.
Reflections on the nature of discovery in science
My autophagy research has always been driven by nothing more than intellectual
curiosity and a thirst to get a better understanding of life through protein dynamics within the cell. When I started my work, I never thought it would become
relevant in diseases as diverse as neuro-degeneration, infectious disease, cancer
and others in such a short time. But now autophagy research has become a major
field in biology. Figure 25 shows that the number of papers related to autophagy
published in the literature continues to rapidly increase. It goes without saying
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FIGURE 24. Diagram of autophagy-dependent RNA degradation in yeast. The standard
process of RNA degradation by autophagy is indicated by thick arrows, whereas minor
products are indicated by thin arrows.
that this is thanks in large part to the tremendous efforts of many researchers
all over the world, so I would like to express my sincere thanks and share this
honour with all of them.
But Figure 25 also tells us it takes a while to establish a new field. Truly original discoveries in science are often triggered by unpredictable and unforeseen
small findings. Nowadays, the distance between basic discoveries and practical
applications is getting closer. While this is exciting, scientists are increasingly
required to provide evidence of immediate and tangible applications of their
work. It is my sincere hope that society is able to nurture not only purposeoriented science, but also science as a core cultural activity. If my small idea
and decades of work have made a contribution to fundamental science through
autophagy research, and the Nobel Foundation recognizes the basic nature of
this work with this prize, it is honestly my greatest pleasure and satisfaction as
a basic scientist.
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309
FIGURE 25. Number of papers published in the field of autophagy. Major developments
in the Ohsumi lab are indicated in boxes for the relevant years.
Acknowledgements and closing remarks
I feel that I have been very fortunate over the 40 years of my research career. I
am most appreciative for the many wonderful colleagues who I have worked
with over the years. I have been lucky to work with postdoctoral researchers and
graduate students who appreciate the importance of basic research. Many of
them have shared my passion to study difficult projects that do not necessarily
give immediate results. I am very pleased to note that many of these colleagues
are now working at a range of universities as researchers. I have also been very
fortunate to have met and worked with many collaborators over the years who
have helped us along the way. In particular I want to note the contribution of two
researchers who have collaborated closely with us for over a decade in the systematic analysis of the Atg proteins: Nobuo Noda, who now works at the Institute
of Microbial Chemistry, and Fuyuhiko Inagaki, who very unfortunately passed
away in 2016. I deeply appreciate their collaboration over the years.
I have only been able to make these achievements thanks to a good dose of
fortune, the excellent colleagues who have joined me on this journey, the great
efforts of many collaborators, good friends, the continuous support of many
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grants and my caring family, especially my wife Mariko. I am truly moved by
your support over the years.
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